IL-17C is a driver of damaging inflammation during Neisseria gonorrhoeae infection of human Fallopian tube

The human pathogen Neisseria gonorrhoeae ascends into the upper female reproductive tract to cause damaging inflammation within the Fallopian tubes and pelvic inflammatory disease (PID), increasing the risk of infertility and ectopic pregnancy. The loss of ciliated cells from the epithelium is thought to be both a consequence of inflammation and a cause of adverse sequelae. However, the links between infection, inflammation, and ciliated cell extrusion remain unresolved. With the use of ex vivo cultures of human Fallopian tube paired with RNA sequencing we defined the tissue response to gonococcal challenge, identifying cytokine, chemokine, cell adhesion, and apoptosis related transcripts not previously recognized as potentiators of gonococcal PID. Unexpectedly, IL-17C was one of the most highly induced genes. Yet, this cytokine has no previous association with gonococcal infection nor pelvic inflammatory disease and thus it was selected for further characterization. We show that human Fallopian tubes express the IL-17C receptor on the epithelial surface and that treatment with purified IL-17C induces pro-inflammatory cytokine secretion in addition to sloughing of the epithelium and generalized tissue damage. These results demonstrate a previously unrecognized but critical role of IL-17C in the damaging inflammation induced by gonococci in a human explant model of PID.


Sample preparation
To assess membrane-associated proteins in the cell-free supernatant, filtered supernatant (n=1) was ultracentrifuged for 1 hr at 400,000 x g using a TLA-110 rotor.Pelleted outer membrane vesicles were washed and resuspended in 20µl water.Subsequently diluted with 80µl of 1% SDS, 2mM EDTA and 20mM Tris pH 7.5 then sonicated for 3 minutes at room temperature in bath sonicator.Proteins were precipitated with addition of TCA (10% final) and acetone (50% final), washed in cold acetone and finally resuspended and denatured in 30 µl of 8M urea.200 µl total volume trypsin/LysC digestion was carried out next where the samples were first diluted to 120µl final volume with 5μl of 25mM DTT and 85μl 25mM NH4HCO3 (pH8.5) for the reduction step, which was carried out for 15 minutes at 56°C.After cooling on ice to room temperature 6μl of 55mM CAA (chloroacetamide) was added for alkylation where samples were incubated in darkness at room temperature for 15 minutes.This reaction was quenched by 16μl addition of 25mM DTT.Subsequently 10μl of Trypsin/LysC solution [100ng/μl 1:1 Trypsin (Promega):LysC (FujiFilm) mix in 25mM NH4HCO3] along with 48μl of 25mM NH4HCO3 (pH8.5) was added to the samples for a final 200µl volume.Digests were carried out overnight at 37°C then subsequently terminated by acidification with 2.5% TFA [Trifluoroacetic Acid] to 0.3% final.Digests were desalted using Pierce™ C18 SPE pipette tips (100µl volume) per manufacturer protocol and eluted in 20µl of 70/30/0.1% ACN/H2O/TFA, then dried to completion in the speed-vac and finally reconstituted to 1.25µg/µl final concentration in 0.1% formic acid.

LC/GC parameters and MS acquisition settings
Peptides were analyzed by nanoLC-MS/MS using the Agilent 1100 nanoflow system (Agilent) connected to hybrid linear ion trap-orbitrap mass spectrometer (LTQ-Orbitrap Elite™, Thermo Fisher Scientific) equipped with an EASY-Spray™ electrospray source (held at constant 35°C).Chromatography of peptides prior to mass spectral analysis was accomplished using capillary emitter column (PepMap® C18, 3µM, 100Å, 150x0.075mm,Thermo Fisher Scientific) onto which 2µl of extracted peptides was automatically loaded.NanoHPLC system delivered solvents A: 0.1% (v/v) formic acid , and B: 99.9% (v/v) acetonitrile, 0.1% (v/v) formic acid at 0.50 µL/min to load the peptides (over a 30 minute period) and 0.3µl/min to elute peptides directly into the nano-electrospray with gradual gradient from 0% (v/v) B to 30% (v/v) B over 250 minutes followed by rapid gradient from 30% (v/v) B to 50% (v/v) B over 10 minutes and concluded with 7 minute flush-out from 50% (v/v) B to 95% (v/v) B at which time a 2 minute column conditioning at 95% (v/v) B took place.As peptides eluted from the HPLCcolumn/electrospray source survey MS scans were acquired in the Orbitrap with a resolution of 120,000 followed by CID-type MS/MS fragmentation of 30 most intense peptides detected in the MS1 scan from 350 to 1800 m/z; redundancy was limited by dynamic exclusion.

Data annotation/validation/analysis procedures
Elite acquired raw MS/MS data files were converted to mgf file format using MSConvert (ProteoWizard: Open Source Software for Rapid Proteomics Tools Development).Resulting mgf files were used to search against Neisseria gonorrhoeae MS11 (2,208 total entries) concurrently with cRAP common lab contaminant database (116 total entries) using in-house Mascot search engine 2.7.0 [Matrix Science] with fixed Cysteine carbamidomethylation and variable Methionine oxidation plus Asparagine or Glutamine deamidation.Peptide mass tolerance was set at 10 ppm and fragment mass at 0.6 Da.Protein annotations, significance of identification and spectral based quantification was done with Scaffold software (version 5.0.1,Proteome Software Inc., Portland, OR).Peptide identifications were accepted if they could be established at greater than 93.0%probability to achieve an FDR less than 1.0% by the Scaffold Local FDR algorithm.Protein identifications were accepted if they could achieve an FDR less than 1.0% and contained at least 2 identified peptides.Protein probabilities were assigned by the Protein Prophet algorithm (1).Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.Proteins sharing significant peptide evidence were grouped into clusters.Sequences were analyzed using SignalP 6.0 to predict subcellular localization (2).Supplementary Table 1.Proteins in N. gonorrhoeae outer membrane vesicle preparations from cell-free supernatant.Fifteen most abundant proteins as determined by total spectral count.Cell-free supernatant was ultracentrifuged at 400,000 x g and the pelleted material was digested with try trypsin before mass spectrometry analysis.Sequences were run through SignalP 6.0 to determine subcellular localization.SPI and SPII proteins are transported by the Sec pathway.SPI is cleaved by signal peptidase I, SPII is cleaved by signal peptidase II and predicted to be a lipoprotein.Cyt proteins are not predicted to have signal peptides and thus assumed to be cytoplasmic.Source data are provided in the Source Data file.n = 1

Supplementary Tables and Figures
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2 FCSupplementary Figure 1 .
Transcriptional analysis of 24h N. gonorrhoeae supernatant treated human Fallopian tube tissues.A) Volcano plot of all genes expressed in mock and 24h N. gonorrhoeae supernatant treated tissues.Each dot represents one gene.Those colored black represent genes with a false discovery rate (FDR) > 0.05 while blue dots represent those with an FDR < 0.05.Dots below the gray line represent genes with p > 0.05 (-log10(p-value) < 1.3) while those above the line represent those with p < 0.05.Genes to the left of the red line are repressed 2-fold or greater (log2FC ≥ 1).Genes to the right of the green line are induced 2-fold or greater."FC" = fold change.B) Relative expression levels (log2FC) of genes significantly different (FDR < 0.05, p < 0.05, log2FC ≥ 1) between treatments.All significantly repressed genes (log2FC ≤ -1) are displayed.Only the top 20 significantly induced genes (log2FC ≥ 1) are displayed.Blue bar color is indicative of assignment to a PANTHER, GO, or KEGG inflammation pathway.For A and B, the posterior probability of differential expression and the FDR estimate for multiple comparisons were determined by a Bayesian model (EBSeq).C) PANTHER pathway analysis.The number within each slice is the number of significantly different genes assigned to the pathway.D) Gene ontology (GO) biological function pathway analysis, top 15 pathways.Source data are provided in the Source Data file.
signaling pathway Pos.reg. of ERK1 and ERK2 cascade Detoxification of copper ion Negative regulation of growth Cellular response to zinc ion Cellular response to copper ion Cellular zinc ion homeostasis Lymphocyte chemotaxis Cellular response to cadmium ion Cellular response to interferon-gamma Cellular response to tumor necrosis factor